Application Note: Cleaning Protocols to Reduce Effects of Pressure Rise Over the Lifetime of a GORE™ Protein Capture Devices

United States

Two cleaning protocols outlined in this application note potentially extend the lifetime of a GORE Protein Capture Device in the event that pressure elevates over the expected lifetime of the device.


Objective

Provide two cleaning protocols to implement in the event that a GORE Protein Capture Device with Protein A experiences pressure rise over the course of the device lifetime. 

Purpose

The GORE Protein Capture Device improves potential processing capabilities for affinity Protein A chromatography, thus shortening the time needed to purify a given monoclonal antibody. Increased speed and capacity allows for more purification cycles in a given amount of time. Therefore, the lifetime of a device might be reached more rapidly than Protein A resins with less capacity and slower speeds to purification. Two cleaning protocols outlined in this application note potentially extend the lifetime of a GORE Protein Capture Device in the event that pressure elevates over the expected lifetime of the device. 

Materials/Equipment

  • Liquid Chromatography System (LC System)
  • PROA101 GORE Protein Capture Devices (3)
  • Chemicals outlined in recommended protocols
  • Operating Instructions for GORE Protein Capture Devices

Protocols

Three GORE Protein Capture Devices (PROA101) were used to perform CHO Cell Harvest purifications until pressure rises were observed. Separate cleaning protocols were implemented for each device to reduce the observed pressure rise. Tables 1 and 2 outline the steps and buffers used for both cleaning protocols.

Buffer Column Volumes
(CV)
Flow Rate
(mL/min)
Volume
(mL)
Step Process
PBS* 10.0 3.0 30.0 Equilibration
Feed Stock **** 3.0 **** Load
PBS 10.0 3.0 30.0 Load Wash
Citrate** 12.5 3.0 37.5 Elution
DTT Cleaning Buffer*** 10.0 1.0 10.0 Enhanced Clean
DTT Hold **** 10 Minute Hold Step
PBS 10.0 3.0 30.0 Cleaning Wash
0.2M NaOH 3.6 1.2 4.3 CIP
PBS 10.0 3.0 30.0 Equilibration

* PBS Phosphate-buffered saline (150 mM NaCl, 50 mM Phosphate, pH 7.4)
** Citrate (100 mM Citrate, pH 3.4)
*** DTT Cleaning Buffer (50 mM Tris Base, 100 mM NaCl, 1% SDS, 10-15 mM 1,4-Dithiothreitol (DTT), pH 10.4, Conductivity 11-12 mS/cm)
**** DTT Hold (static reduction step)
***** Feed load dependent

Buffer Column Volumes
(CV)
Flow Rate
(mL/min)
Volume
(mL)
Step Process
L-Arginine HCl* 5.0 3.0 15.0 Acidic Equilibration
DTT Cleaning Buffer** 10.0 1.0 10.0 Enhanced Clean
DTT Hold*** 10 Minute Hold Step
L-Arginine HCl 10.0 3.0 30.0 Acidic Wash
02M NaOH 3.6 1.2 4.3 CIP
PBS**** 10.0 3.0 30.0 Equilibration

* L-Arginine HCl (100 mM L-Arginine HCl, pH 2.3)
** DTT Cleaning Buffer (50 mM Tris Base, 100 mM NaCl, 1% SDS, 10-15 mM DTT, pH 10.4, Conductivity 11-12 mS/cm)
*** DTT Hold (static reduction step)
**** PBS Phosphate-buffered saline (150 mM NaCl, 50 mM Phosphate, pH 7.4)

The first cleaning protocol outlined in Table 1, Intra-Cycle Cleaning protocol, was implemented while performing feed cycling& experiments during a mid-cycle Dynamic Binding Capacity (DBC)test as defined in the operating instructions.

The second cleaning protocol outlined in Table 2 was implemented as a standalone cycle, independent of any purification cycling or DBC tests as pressure approached the maximum recommended operating value after performing multiple cycles with a CHO Cell Harvest.

All CHO Cell Harvest purifications followed the operating instructions for GORE Protein Capture Devices. The metric used to observe column pressure rise over the course of use was delta column pressure (dP) (output from the LC unit). Specifically, maximum loading delta pressure in the presence of either human monoclonal antibody (CHO Cell Harvest), polyclonal human IgG (DBC evaluations), or initial PBS delta column pressure at the beginning of each purification cycle was used.

Results

Intra-Cycle Method

While performing multiple CHO Cell Harvest purifications with harvest titer ranges from ≤ 0.50 mg/mL to 2.0 mg/mL, significant pressure rise was observed for both devices. During the cycling, a DBC test was performed with polyclonal hIgG and implementation of the intra-cycle cleaning protocol.

Figure 1 indicates the delta column pressure (purple line) during the intra-cycle cleaning protocol performed during the DBC test from the PROA101 device. An observed spike in both pressure and UV (blue line) can be observed during the DTT cleaning step as well as the subsequent PBS wash after the 10 minute static reduction step. The subsequent PBS equilibration performed at the end of this intra-cycle cleaning demonstrated a lower delta column pressure than the pressure at the beginning of the cycle.

Figure 2 shows a second DBC performed with the same device after the intra-cycle DTT cleaning protocol (orange line) compared to the run shown in Figure 1 prior to cleaning.

Figure 3 demonstrates a recovery in DBC after the Intra-Cycle DTT cleaning protocol (orange line) compared to DBC from Figure 1 prior to cleaning.

Figure 1 graph showing intra-cycle DTT washing protocol with pressure and UV absorbance

Figure 1: Intra-Cycle (DTT) Washing Protocol with delta column pressure (MPa) in purple, and UV absorbance (280nm) in blue.

Figure 2 graph showing pressure curves from follow-up DBC after intra-cycle DTT washing protocol

Figure 2: Pressure Curves from the Follow-up DBC (orange line) after performing Intra-Cycle (DTT) Washing Protocol (blue line).

Figure 3 graph showing UV trace of DBC before and after intra-cycle DTT washing protocol

Figure 3: UV Trace of DBC showing the difference before performing the Intra-Cycle (DTT) washing protocol (blue line) vs. after the washing protocol (orange line).

A second device was tested using the intra-cycle cleaning protocol as shown in Table 3. The maximum loading delta column pressure in the presence of either human monoclonal antibody (CHO Cell Harvest) or polyclonal human IgG (DBC evaluations) throughout cycling and including the implementation of the intra-cycle cleaning protocol at cycle 12 is shown in Table 3 and Figure 4.

Protein Purified Cycle Max load dP (MPa)
Polyclonal hIgG DBC 1 0.186
CHO Harvest Purification 2 0.207
CHO Harvest Purification 3 0.212
CHO Harvest Purification 4 0.216
CHO Harvest Purification 5 0.219
CHO Harvest Purification 6 0.222
CHO Harvest Purification 7 0.228
CHO Harvest Purification 8 0.233
CHO Harvest Purification 9 0.241
CHO Harvest Purification 10 0.249
CHO Harvest Purification 11 0.255
Polyclonal hIgG DBC* 12 0.265
Polyclonal hIgG DBC** 13 0.193

* Prior to Intra-Cycle DTT Wash Protocol
** DBC Post Intra-Cycle DTT Cleaning Protocol

Figure 4: Graph showing maximum loading delta column pressure and recovery

Figure 4: Maximum Loading Delta Column Pressure during CHO Cell Harvest Purifications along with Subsequent Pressure Recovery after implementing the Intra-Cycle DTT Washing Protocol.

Inter-Cycle Method

A third device was cycled with CHO cell feed until the delta column pressure rose to near maximum recommended operating values (0.4MPa). The device was subsequently cleaned using the inter-cycle cleaning protocol. This cleaning protocol was implemented independently of feed purifications and/or DBC testing.

Figure 5 shows the UV chromatogram (280nm) for the inter-cycle DTT wash protocol. A significant UV absorbance peak is observed when exposed to the DTT cleaning solution as well as when followed up with the acidic wash buffer. This inter-cycle DTT washing protocol recovered most of the pressure rise observed in the device over the course of cycling as shown in Figure 6.

Figure 6 indicates the maximum delta column pressure during the initial PBS equilibration step for each purification cycle. The device underwent 30 purification cycles followed by the inter-cycle washing protocol. The inter-cycle cleaning method restored column pressure to levels well within the recommended operating pressure.

Figure 5 graph showing UV trace from inter-cycle DTT washing protocol

Figure 5: UV Trace from Inter-Cycle DTT Washing Protocol.

Figure 6 graph showing equilibration delta column pressure and subsequent recovery after washing protocol

Figure 6. Maximum Initial PBS Equilibration Delta Column Pressure during CHO Cell Harvest Purifications along with Subsequent Pressure Recovery after implementing the Inter-Cycle DTT Washing Protocol.

Conclusion

Two washing protocols were explored using DTT– both of which successfully reduced the delta column pressures of (3) PROA101 devices after delta column pressures had increased over the course of use.

The three PROA101 devices spanned CHO Cell Harvest titer ranges from ≤0.50 mg/mL to 2.0 mg/mL and all experienced different levels of pressure rise over the course of cycling. The pressure rise experienced for all three of the devices used were reduced once cleaned with one of the two DTT washing protocols.

These two cleaning protocols provide options for the effective recovery of unexpected delta column pressure rise during the use of a GORE Protein Capture Device.

Gore PharmBIO Products

Our technologies, capabilities, and competencies in fluoropolymer science are focused on satisfying the evolving product, regulatory, and quality needs of pharmaceutical and bioprocessing customers, and medical device manufacturers. GORE™ Protein Capture Devices, like all products in the Gore PharmBIO Products portfolio, are tested and manufactured under stringent quality systems. These high-performance products provide creative solutions to our customers’ design, manufacturing, and performance-in-use needs.


GORE Protein Capture Devices are intended for research use only and should not be used for clinical or diagnostic procedures. All technical information and advice given here is based on our previous experiences and/or test results. We give this information to the best of our knowledge, but assume no legal responsibility. Customers are asked to check the suitability and usability of our products in the specific applications, since the performance of the product can only be judged when all necessary operating data is available. Gore’s terms and conditions of sales apply to the purchase and sale of the product.